A new gymnophallid trematode from the intestine of mice infected with metacercariae from the razor clam Sinonovacula constricta

Meiogymnophallus sinonovaculae n. sp. (Digenea: Gymnophallidae) is described from metacercariae found in the razor clam, Sinonovacula constricta, in the Republic of Korea, and adults recovered from the small intestine of experimentally infected mice. The worms are characterized by paired clusters of vitelline follicles, a well-developed pars prostatica with abundant prostate cells, a voluminous undivided seminal vesicle (in adults and metacercariae), and large, grouped, domelike sensory papillae on the ventral surface anterior to the ventral sucker (in metacercariae). This new species resembles the type species Meiogymnophallus affinis, but differs in having compact, elliptical, and 4-5-lobed vitellaria, and an excretory vesicle with bicornuated anterior arms reaching to the oral sucker. This is the second documentation of the presence of a species of Meiogymnophallus in the Republic of Korea.     

Populations genetic structure of the razor clam Sinonovacula constricta from China,Korea and Vietnam

The population genetic structure of the razor clam Sinonovacula constricta was investigated between populations collected from China, Korea and Vietnam using cytochrome oxidase subunit 1 (COI) mtDNA and internal transcribed spacer 2 (ITS2) rDNA markers. A total of 622 bp of COI and 495 bp of ITS2 were sequenced. Highly significant Fst values and low rates of migration were observed from populations between Vietnam and China, (Fst = 0.7578, P < 0.001, Nm = 0.05 for COI; Fst = 0.91389, P < 0.001, Nm = 0.07 for ITS2) and Korea (Fst = 0.79783, P < 0.001, Nm = 0.07 for COI; Fst = 0.74143, P < 0.001, Nm = 0.03 for ITS2). However, lower Fst values and higher gene flow were detected between populations from China and Korea (Fst = 0.25733, P < 0.001, Nm = 0.73) based on COI analysis. The similar pattern was also captured by ITS2 rDNA marker. In addition, Neighbor-joining phylogenetic tree analysis of the two markers resulted in one cluster consisting of haplotypes from Vietnam, and a second group comprising haplotypes from China and Korea. This research revealed a closer genetic relationship between clam population from China and Korea but less similarity to population from Vietnam.     

Chromosome‐level genome assembly of the razor clam Sinonovacula constricta (Lamarck, 1818)

Bivalves, a highly diverse and the most evolutionarily successful class of invertebrates native to aquatic habitats, provide valuable molecular resources for understanding the evolutionary adaptation and aquatic ecology. Here, we reported a high‐quality chromosome‐level genome assembly of the razor clam Sinonovacula constricta using Pacific Bioscience single‐molecule real‐time sequencing, Illumina paired‐end sequencing, 10X Genomics linked‐reads and Hi‐C reads. The genome size was 1,220.85 Mb, containing scaffold N50 of 65.93 Mb and contig N50 of 976.94 Kb. A total of 899 complete (91.92%) and seven partial (0.72%) matches of the 978 metazoa Benchmarking Universal Single‐Copy Orthologs were determined in this genome assembly. And Hi‐C scaffolding of the genome resulted in 19 pseudochromosomes. A total of 28,594 protein‐coding genes were predicted in the S. constricta genome, of which 25,413 genes (88.88%) were functionally annotated. In addition, 39.79% of the assembled genome was composed of repetitive sequences, and 4,372 noncoding RNAs were identified. The enrichment analyses of the significantly expanded and contracted genes suggested an evolutionary adaptation of S. constricta to highly stressful living environments. In summary, the genomic resources generated in this work not only provide a valuable reference genome for investigating the molecular mechanisms of S. constricta biological functions and evolutionary adaptation, but also facilitate its genetic improvement and disease treatment. Meanwhile, the obtained genome greatly improves our understanding of the genetics of molluscs and their comparative evolution.     

Identification and characterization of a clam ferritin from Sinonovacula constricta

Ferritin, a major iron storage protein of most living organisms, plays a crucial role in iron metabolism. Here we reported the isolation and characterization of a cDNA of ferritin gene from Sinonovacula constricta (denoted as ScFER). The full-length cDNA of ScFER was of 996 bp, consisting of a 5'-UTR of 120 bp, a 3'-UTR of 360 bp, and a complete open reading frame of 516 bp encoding a polypeptide with 171 amino acid residues. The predicted molecular mass of deduced amino acid of ScFER was 19.76 kDa and the theoretical pI was 5.07. Quantitative real-time PCR was employed to analyze the expression profiles of ScFER mRNA in muscle, mantle and visceral mass after iron exposure. The peak expression level of ScFER in the three tissues was 1.79-fold, 1.31-fold and 3.51-fold increases in muscle, mantle and visceral mass, respectively. The polyclonal antibodies generated from the recombinant product of ScFER could be specifically identified not only the recombinant product, but also the native protein from muscle. All these results strongly suggested that ScFER was involved in the iron metabolism regulation in S. constricta.     

Development of microsatellite markers in Qualea grandiflora (Vochysiaceae) and transferability to congeneric species,typical trees of the Brazilian savanna

This study aimed to isolate and characterize microsatellite markers for Qualea grandiflora and to test their transferability to congeneric species Qualea multiflora and Qualea parviflora. These three species are widespread in the Cerrado, the largest, richest and probably the most threatened tropical savanna in the world. We characterized ten markers in 40 individuals belonging to two populations of Q. grandiflora and eight markers in 20 individuals belonging to one population of Q. multiflora and Q. parviflora. In Q. grandiflora, considering all 40 analyzed individuals, the number of alleles per locus ranged from eight to 21, and the average was 11.60. The mean number of alleles per locus was 8.8 and 7.3 in each population. The observed and expected heterozygosities (H_o and H_e) within populations varied from 0.235 to 0.944 and from 0.225 to 0.932, respectively. In Q. multiflora the number of alleles varied from two to 11 with an average of 5.75; the H_o ranged from 0.150 to 0.950, while H_e ranged from 0.191 to 0.817. In Q. parviflora, considering the seven polymorphic loci, the number of alleles ranged from two to 13, with an average of 7.5, while H_o ranged from 0.211 to 0.944, and H_e ranged from 0.193 to 0.906. The polymorphism level of the microsatellite markers here described enable them as powerful tools for future population genetic studies in these species, helping to answer ecological and evolutionary questions.     

Polymorphisms of LAP3 gene and their association with the growth traits in the razor clam Sinonovacula constricta

Molecular Biology Reports - Leucine aminopeptidase 3 (LAP3) is an important proteolytic enzyme that catalyzes the hydrolysis of leucine residues from the amino termini of protein or peptide...     

Development and characterization of new microsatellite loci from Chinese mitten crab (Eriocheir sinensis)

A set of new microsatellite loci were isolated from Chinese mitten crab and characterized in two Chinese mitten crab populations. The number of the alleles ranged from 3 to 8 and observed and expected heterozygosity varied from 0.0870 to 0.9565 and 0.3092 to 0.8367 in each population, respectively. Loci HLJEsin1, HLJEsin4, HLJEsin30 in CLN population and HLJEsin6 in CJS population presented significant deviation from Hardy–Weinberg equilibrium (PHWE < 0.001). These markers will be useful for population studies.     

Isolation and characterization of polymorphic microsatellite loci in the razor clam Ensis siliqua

Five polymorphic microsatellite loci in the razor clam Ensis siliqua are described. A collection consisting of 34 individuals from Finisterre, Spain, was analysed. Loci were isolated from the sequences of intersimple sequence repeat (ISSR) markers. Detailed analysis of 42 ISSR markers led to the design of 16 primer pairs. Five of these yielded consistent and polymorphic products. The number of alleles ranged from five to 23 per locus with the observed heterozygosity ranging from 0.46 to 0.94. Linkage equilibrium was observed in all loci and three of them showed significant deviations from Hardy–Weinberg equilibrium.     

Isolation and characterization of microsatellite loci in the red mangrove Rhizophora mangle (Rhizophoraceae) and its related species

Fourteen microsatellite markers were isolated from the red mangrove Rhizophora mangle (Rhizophoraceae), a widely distributed mangrove plant in the New World and West Africa. The range of expected heterozygosity of these markers was 0.000–0.672 in the two populations of R. mangle. Cross-species testing was examined for five other species of Rhizophora, and Kandelia candel and Bruguiera gymnorrhiza. All 14 markers could be amplified in R. samoensis, thirteen in R. racemosa, and six markers in all other species of Rhizophora. Our findings greatly increase the utility of these markers.     

Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: Characterization of four fatty acyl elongases and a novel desaturase capacity

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the ‘Sprecher pathway’. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs.     

The razor clam Sinonovacula constricta uses the strategy of conversion of toxic ammonia to glutamine in response to high environmental ammonia exposure

Molecular Biology Reports - High ammonia can inhibit the survival and growth, and even cause mortality of razor clam (S. constricta). The accumulation of ammonia to lethal concentrations in some...     

Characteristics and transferability of new apple EST-derived SSRs to other Rosaceae species

Genic microsatellites or simple sequence repeat markers derived from expressed sequence tags (ESTs), referred to as EST–SSRs, are inexpensive to develop, represent transcribed genes, and often have assigned putative function. The large apple (Malus × domestica) EST database (over 300,000 sequences) provides a valuable resource for developing well-characterized DNA molecular markers. In this study, we have investigated the level of transferability of 68 apple EST–SSRs in 50 individual members of the Rosaceae family, representing three genera and 14 species. These representatives included pear (Pyrus communis), apricot (Prunus armeniaca), European plum (P. domestica), Japanese plum (P. salicina), almond (P. dulcis), peach (P. persica), sour cherry (P. cerasus), sweet cherry (P. avium), strawberry (Fragaria vesca, F. moschata, F. virginiana, F. nipponica, and F. pentaphylla), and rose (Rosa hybrida). All 68 primer pairs gave an amplification product when tested on eight apple cultivars, and for most, the genomic DNA-derived amplification product matched the expected size based on EST (in silico) data. When tested across members of the Rosaceae, 75% of these primer pairs produced amplification products. Transferability of apple EST–SSRs across the Rosaceae ranged from 25% in apricot to 59% in the closely related pear. Besides pear, the highest transferability of these apple EST–SSRs, at the genus level, was observed for strawberry and peach/almond, 49 and 38%, respectively. Three markers amplified in at least one genotype within all tested species, while eight additional markers amplified in all species, except for cherry. These 11 markers are deemed good candidates for a widely transferable Rosaceae marker set provided their level of polymorphism is adequate. Overall, these findings suggest that transferability of apple EST–SSRs across Rosaceae is varied, yet valuable, thereby providing additional markers for comparative mapping and for carrying out evolutionary studies.     

Characterization of nine novel microsatellite loci for the Venus clam (Cyclina sinensis)

The Venus clam, Cyclina sinensis, is one of the most important bivalves in China marine aquaculture. Using (CA)(15)-enriched genomic libraries of this species, nine novel polymorphic microsatellite loci were isolated and characterized. The mean number of observed alleles per locus was 16 (range 8-24). The observed and expected heterozygosity ranged from 0.119 to 0.872 and from 0.626 to 0.931, respectively. Three loci had significant departure from Hardy-Weinberg equilibrium and non-significant linkage disequilibrium was found among all nine loci. These highly informative microsatellite markers should be useful for population genetic analyses of C. sinensis.     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

Characterization of microsatellite loci in Spartina species (Poaceae)

The cordgrasses in the genus Spartina have become model organisms for studying biological invasions from both ecological and genetic perspectives. Here we characterize 11 disomic loci in Spartina alterniflora that show promise for population studies and for studying hybridization events between S. alterniflora and S. foliosa. Comparisons among invasive and native S. alterniflora populations showed that levels of allelic variation are lower in invasive populations. In addition, nearly all loci that amplified in S. foliosa populations and in a swarm of S. alterniflora×foliosa hybrids were polymorphic. We also found that several loci amplified successfully in other Spartina species.     

Characterization of 22 microsatellite marker loci in the Madagascar rousette (Rousettus madagascariensis)

Twenty-two nuclear microsatellite loci were isolated from a genomic DNA library derived from Madagascar’s Rousettus madagascariensis. Marker characteristics were determined from a single population (37 individuals) from Fort Dauphin (southeastern Madagascar). Sixteen of the 22 loci were within Hardy–Weinberg expectations. These loci are highly informative with polymorphic information content values ranging between 0.757 and 0.916. These loci will provide valuable information for the study of population genetics and gene flow within this species of bats. Due to the dramatic reduction and alteration of their habitat, data generated utilizing this marker suite will potentially provide additional information for the effective long-term management of this near-threatened bat species.     

Characterization of 21 microsatellite marker loci in the silky sifaka (Propithecus candidus)

The silky sifaka, Propithecus candidus, considered one of the rarest and most endangered primates in the world, exists in only a few fragmented forests in northeastern Madagascar. This species faces increasing pressures as a direct result from loss of habitat in the form of tavy (slash and burn agriculture), illegal logging and mining along with hunting for subsistence, even within protected areas. We report a marker suite of 21 loci developed from genomic DNA from a silky sifaka collected in 2003 from Anjanaharibe-Sud Special Reserve. Polymorphism of each locus evaluated in 18 individuals pooled from Marojejy National Park and Anjanaharibe-Sud Special Reserve. The number of observed alleles per locus ranges from 2 to 7. The observed and expected heterozygosity were 0.389–0.889 and 0.322–0.789, respectively. The information revealed in this study will provide useful tools for further study of the social structure and population dynamics of the silky sifaka to facilitate conservation management in the imminent future.